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Research Letters |

Serologic Markers of Lyme Disease in Children With Autism FREE

Mary Ajamian, MS; Barry E. Kosofsky, MD, PhD; Gary P. Wormser, MD; Anjali M. Rajadhyaksha, PhD; Armin Alaedini, PhD
[+] Author Affiliations

Author Affiliations: Department of Medicine, Columbia University Medical Center, New York, New York (Ms Ajamian and Dr Alaedini) (aa819@columbia.edu); Department of Pediatrics, Weill Cornell Medical College, New York, New York (Drs Kosofsky and Rajadhyaksha); and Division of Infectious Diseases, New York Medical College, Valhalla (Dr Wormser).


Letters Section Editor: Jody W. Zylke, MD, Senior Editor.

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JAMA. 2013;309(17):1771-1773. doi:10.1001/jama.2013.618.
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To the Editor: A proposed link between Lyme disease and autism has garnered considerable attention.1,2 Among individuals with autism spectrum disorders, rates of seropositivity for Lyme disease of greater than 20% have been reported.1 However, controlled studies to assess serological evidence of infection with Borrelia burgdorferi (the causative agent of Lyme disease) in patients with autism are lacking.

Serological evidence of infection with B burgdorferi is essential for diagnosing Lyme disease, except in cases of typical erythema migrans skin lesions. To evaluate the suggestion that autism is commonly linked to Lyme disease, we performed Lyme disease serological testing on serum samples from children with autism and those without autism.

Serum samples from 120 children aged 2 through 18 years with autism and those without autism were acquired from the Autism Genetic Resource Exchange (AGRE) (37 with autism and 27 unaffected siblings) and the Weill Cornell Autism Research Program (WCARP) (33 with autism, 8 unaffected siblings, and 15 unrelated healthy controls). All WCARP and some unselected AGRE sites collected serum samples; all available serum samples were included.

Patients from the AGRE program met diagnostic criteria for autism based on both the Autism Diagnostic Observation Schedule (ADOS) and the Autism Diagnostic Interview, Revised, whereas WCARP patients met criteria for autism based only on the ADOS. Participants in the AGRE program have been recruited primarily from the northeastern and western United States; serum samples for this study were collected from August 31, 1999, through April 25, 2001.

The WCARP serum samples were from participants who resided primarily in Connecticut, New Jersey, and New York, and were collected from May 19, 2010, through March 7, 2012. Screening questionnaires were used to evaluate the general health of unrelated controls.

Written informed consent was obtained for all study participants from a parent or guardian. Serum samples from 2 patients with culture-confirmed early Lyme disease were used as positive controls. Specimens were kept at −80°C to maintain stability. This study was approved by the institutional review board of Columbia University Medical Center.

Testing for antibodies to B burgdorferi was performed according to the 2-tier algorithm recommended by the US Centers for Disease Control and Prevention.3 Initial screening for anti– Bburgdorferi immunoglobulin G and M antibodies was performed with separate enzyme-linked immunosorbent assays (ELISAs), according to the manufacturer's protocols (Euroimmun). Specimens classified as borderline or positive were further tested by Western blotting for IgG or IgM antibodies to electrophoresis-separated B burgdorferi strain B31 proteins (Euroimmun).4

Assuming 1% or lower seroprevalence in controls, and at least 20% seroprevalence in cases as suggested, the sample size in this study would provide greater than 90% power with an α level of .05. Differences between groups were analyzed using the 2-tailed Fisher exact test; P values of less than .05 were considered to be statistically significant. Binomial distribution confidence intervals were determined by the Clopper-Pearson exact method.

Seventy children with autism (58 male; mean [SD] age, 7.2 [3.6] years) and 50 unaffected controls (32 male; mean age, 9.0 [4.0] years) were included. Of the patients with autism, 1 was positive by ELISA for anti– B burgdorferi IgG, whereas 4 were borderline by ELISA for IgM. Of the 50 children in the unaffected control group, 4 were positive and 1 was borderline for IgG by ELISA, whereas 1 was positive by ELISA for IgM.

All serum samples that were positive or borderline by ELISA were further analyzed using Western blot and were found to be negative for anti– B burgdorferi antibody reactivity (Table 1 and Table 2). The 95% confidence interval for seroprevalence in children with autism and in unaffected controls was 0% to 5.1%.

Table Graphic Jump LocationTable 1. Serum Immunoglobulin G Antibody Reactivity to Borrelia burgdorferi Protein Bands as Determined by Western Blotting in Patients and Controls Who Were Positive or Borderline for IgG by Enzyme-Linked Immunosorbent Assaya
Table Graphic Jump LocationTable 2. Serum Immunoglobulin M Antibody Reactivity to Borrelia burgdorferi Protein Bands as Determined by Western Blotting in Patients and Controls Who Were Positive or Borderline for IgM by Enzyme-Linked Immunosorbent Assaya

None of the children with autism or unaffected controls had serological evidence of Lyme disease by 2-tier testing. A potential limitation of this study is the lack of information about lifestyle for patients and controls, including time spent outdoors.

The data do not address whether Lyme disease may cause autism-like behavioral deficits in some cases. However, the study's sample size is large enough to effectively rule out the suggested high rates of Lyme disease or associated seroprevalence among affected children.

Author Contributions: Dr Alaedini had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Alaedini.

Acquisition of data: Ajamian.

Analysis and interpretation of data: Ajamian, Kosofsky, Wormser, Rajadhyaksha, Alaedini.

Drafting of the manuscript: Ajamian, Alaedini.

Critical revision of the manuscript for important intellectual content: Ajamian, Kosofsky, Wormser, Rajadhyaksha, Alaedini.

Statistical analysis: Ajamian, Alaedini.

Obtained funding: Alaedini.

Administrative, technical, or material support: Ajamian, Kosofsky, Wormser, Rajadhyaksha, Alaedini.

Study supervision: Alaedini.

Conflict of Interest Disclosures: The authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Dr Wormser reported receiving grants from the Centers for Disease Control and Prevention, the National Institutes of Health, Immunetics Inc, Bio-Rad, DiaSorin Inc, and BioMerieux for research related to Lyme disease; holding stock in Abbott; providing expert witness testimony in malpractice cases involving Lyme disease; serving as unpaid board member at the American Lyme Disease Foundation; serving as an expert witness regarding Lyme disease in a disciplinary action for the Missouri Board of Registration for the Healing Arts; serving as a consultant to Baxter for Lyme vaccine development; and receiving reimbursement for travel expenses from the American Society for Microbiology. Dr Rajadhyaksha reported receiving a grant from The Hartwell Foundation for research related to autism. Dr Alaedini reported receiving grants from the National Institutes of Health, the Department of Defense, and the Lyme Research Alliance for research related to Lyme disease or autism. No other disclosures were reported.

Funding/Support: This study was supported in part by grant W81XWH 10-1-0887 from the Department of Defense and grant 1R56 AI093763-01 from the National Institutes of Health (awarded to Dr Alaedini). The Autism Genetic Resource Exchange (AGRE) is a program of Autism Speaks and is supported, in part, by grant 1U24MH081810 from the National Institute of Mental Health (awarded to Clara M. Lajonchere). The Weill Cornell Autism Research Program (WCARP) is supported in part through funding from the Clinical and Translational Science Center of the Weill Cornell Medical College and by National Institutes of Health grant UL1 TR000457-06.

Role of the Sponsors: The funding agencies had no role in the design and conduct of the study; in the collection, analysis, and interpretation of the data; or in the preparation, review, or approval of the manuscript.

Additional Contributions: We thank Nga M. Lau, MD (Department of Medicine, Columbia University Medical Center), Joseph J. Higgins, MD (Department of Pediatrics, Weill Cornell Medical College), Mary J. Ward, PhD (Department of Pediatrics, Weill Cornell Medical College), Peter H. Green, MD (Department of Medicine, Columbia University Medical Center), and Kaleb Yohay, MD (Department of Pediatrics, Weill Cornell Medical College) for their involvement in recruitment or clinical assessment of study participants. We thank the Weill Cornell Autism Research Program (WCARP) and the participating WCARP families. We thank the Autism Genetic Resource Exchange (AGRE) Consortium and the participating AGRE families. No compensation was received by any of these individuals.

Bransfield RC, Wulfman JS, Harvey WT, Usman AI. The association between tick-borne infections, Lyme borreliosis and autism spectrum disorders.  Med Hypotheses. 2008;70(5):967-974
PubMed   |  Link to Article
Kuhn M, Grave S, Bransfield R, Harris S. Long term antibiotic therapy may be an effective treatment for children co-morbid with Lyme disease and autism spectrum disorder.  Med Hypotheses. 2012;78(5):606-615
PubMed   |  Link to Article
Centers for Disease Control and Prevention (CDC).  Recommendations for test performance and interpretation from the Second National Conference on Serologic Diagnosis of Lyme Disease.  MMWR Morb Mortal Wkly Rep. 1995;44(31):590-591
PubMed
Chandra A, Latov N, Wormser GP, Marques AR, Alaedini A. Epitope mapping of antibodies to VlsE protein of Borrelia burgdorferi in post-Lyme disease syndrome.  Clin Immunol. 2011;141(1):103-110
PubMed   |  Link to Article

Figures

Tables

Table Graphic Jump LocationTable 1. Serum Immunoglobulin G Antibody Reactivity to Borrelia burgdorferi Protein Bands as Determined by Western Blotting in Patients and Controls Who Were Positive or Borderline for IgG by Enzyme-Linked Immunosorbent Assaya
Table Graphic Jump LocationTable 2. Serum Immunoglobulin M Antibody Reactivity to Borrelia burgdorferi Protein Bands as Determined by Western Blotting in Patients and Controls Who Were Positive or Borderline for IgM by Enzyme-Linked Immunosorbent Assaya

References

Bransfield RC, Wulfman JS, Harvey WT, Usman AI. The association between tick-borne infections, Lyme borreliosis and autism spectrum disorders.  Med Hypotheses. 2008;70(5):967-974
PubMed   |  Link to Article
Kuhn M, Grave S, Bransfield R, Harris S. Long term antibiotic therapy may be an effective treatment for children co-morbid with Lyme disease and autism spectrum disorder.  Med Hypotheses. 2012;78(5):606-615
PubMed   |  Link to Article
Centers for Disease Control and Prevention (CDC).  Recommendations for test performance and interpretation from the Second National Conference on Serologic Diagnosis of Lyme Disease.  MMWR Morb Mortal Wkly Rep. 1995;44(31):590-591
PubMed
Chandra A, Latov N, Wormser GP, Marques AR, Alaedini A. Epitope mapping of antibodies to VlsE protein of Borrelia burgdorferi in post-Lyme disease syndrome.  Clin Immunol. 2011;141(1):103-110
PubMed   |  Link to Article
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